We are interested in determining the mechanisms by which human cells control spontaneous and induced mutation rates. In animal cells, DNA synthesis is performed by five distinct classes of DNA polymerases, alpha, beta, delta, epsilon and gamma. We are characterizing the accuracy of DNA synthesis by each of these enzymes in an attempt to understand how mutation rates are controlled. A major focus during the past year has been examination of the fidelity of the three putative replicative DNA polymerases, alpha, beta, delta, and epsilon as well as to examine the effects of replication accessory proteins on fidelity. Accomplishments include a determination of the detailed error specificity of the catalytic subunit of DNA polymerase a from yeast, alone and with several accessory proteins. We have examined the error specificity of DNA polymerases epsilon, and delta, which contain associated exonuclease activity, to determine the contribution of base-selectivity and proofreading to fidelity. While DNA synthesis by purified DNA polymerases in vitro is not accurate enough to account for low spontaneous mutation rates in vivo, actual DNA replication involves the concerted action of a number of pro- teins. We have therefore been examining the fidelity of semiconservative, bidirectional DNA replication by proteins present in extracts of human HeLa cells. Accomplishments this year include a demonstration that replication fidelity is high on both the leading and lagging strands and that exonucleolytic proofreading contributes to fidelity on both strands. These observations are at odds with the current model for a eukaryotic replication fork, which would predict that a difference should exist. We intend to continue these studies to define error rates for each of the four polymerization phases of replication, i.e., origin-specific synthesis, leading and lagging strand synthesis and synthesis associated with RNA primer removal. In order to better understand the effects of known mutagens and carcinogens on the fidelity of DNA synthesis, we intend to extend these types of analyses to DNA substrates that contain defined lesions.